Doom, who outsprinted Norway's Karsten Warholm for the 400m crown on Saturday, produced a stunning finish as Belgium clocked 3min 02.54sec to edge the Americans by six-hundredths.
Lyles, who was beaten into silver by teammate Christian Coleman in the 60m on Friday, was a late inclusion in the relay team with the Paris Olympics in mind, holding his own as he ran a clean third leg.
Chris Bailey was on anchor for the US team, which had led from the gun right through until Doom stole their thunder in the closing metres that had a packed Emirates Arena on their feet.
"The guys put me in the perfect position," said Doom. "I knew there was something possible at the end. I felt a little bit tired after the last few days. But I had a strong kick at the end, so I am really happy."
Lyles justified his late call-up, saying: "To be honest I'd already told people I wanted to run the 4x4 in Paris so I had to prove that, to be on it.
"I came out here and thankfully everybody trusted me to prove that I could do it. I feel like I got the job done."
Lyles added: "Everything I've been doing this year, I've seen a good 4x4 leg, I've seen great 60 metres and I've been able to run three rounds in one day. I'm very positive about Paris."
Jonathan Sacoor, Dylan Borlee and Christian Iguacel embraced their starring teammate Doom as he crossed the line, screaming in delight, as the US quartet also featuring Jacory Patterson and Matthew Boling were left ruing their luck.
The result confirmed Doom as a contender going into the June 7-12 European Championships ahead of the Paris Olympics.
"This means a lot of confidence, not just for me but for the team," Doom said. "It was really a team effort. It is amazing back-to-back gold medal. What a fantastic night."
He admitted after beating hurdles king Warholm with another fast finish that he normally just focused on the relay.
"I loved running individually," he said after clinching individual gold.
"To beat Karsten Warholm is really amazing. He's an Olympic champion and has won almost everything there is. Not many people have beaten him."
US anchorman Bailey added: "Not many people try to get to hold off a world champion, so it is what it is."
